Protonated species are often postulated as key intermediates in enzyme-induced as well as simple chemical reactions. In this research proposal a simple method of some generality is outlined which, when applied to biological reactions, can be used to establish not only the protonated species, but also the specific site of protonation. Irreversibly alkylated, charged analogs of the suspected protonated intermediates are simply synthesized, and then tested for the desired reactivity. Four representative examples of this thesis are examined. The coenzyme, biotin, plays a pivotal role in CO2 fixation reactions, for example in the conversion of acetyl CoA to malonyl CoA. There is considerable controversy concerning the exact nature of the CO2-transfer agent. Using "irreversible acids" we would attempt to ascertain whether or not protonated forms of biotin are needed for this transfer. Flavins play an essential role in the oxidation-reductions that occur in nature. These reactions are not well understood at the molecular level. Protonated flavin intermediates can be envisaged, with the proposed redox reaction occurring by a two electron transfer mechanism. Quinones are important catalysts in biological redox systems. Their effect is usually exerted via electron transfer pathways. A second mechanism, however, involving the chemical generation of an oxenium (protonated) intermediate is also conceivable. A protonated intermediate may be essential in the enzyme catalyzed deamination of deoxycytidine-5'-phosphate. This enzyme, deoxycytidylic acid aminohydrolase, is thought to have a vital role in controlling the concentration of deoxynucleotides in higher organisms.